1. Volume 11, Issue 21, Pages 1645-1655 (October 2001)
Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex 
Sonja Krugmann, Ingrid Jordens, Kris Gevaert, Mariëtte Driessens, Joel Vandekerckhove, Alan Hall 
Current Biology 
Volume 11, Issue 21, Pages (October 2001)
DOI: /S (01)

2. Figure 1
IRSp53 interacts with Cdc42 through a partial CRIB motif. (a) Flag-tagged, constitutively active Rac or Cdc42 alone or together with Myc-tagged IRSp53 were transiently expressed in COS-7 cells. Lysates were subjected to immunoprecipitations using an antibody against the Flag epitope. GTPases and IRSp53 were detected by immunoblot using anti-Flag antibodies and an antiserum raised against IRSp53. (b) Top, a schematic diagram of the IRSp53 domain structure is shown. Below, a panel of IRSp53 truncations, deletions, and point mutations in the partial CRIB motif and SH3 domain are shown. These were designed for expression as GST fusion proteins in E. coli or with an N-terminal Myc or Flag epitope tag in mammalian cells. Tags are indicated by ovals. (c,e) GST fusion proteins, as indicated on the right-hand side, were applied to a nitrocellulose membrane and overlaid with [32P]GTP-loaded L61Rac or L61Cdc42 as indicated above. After extensive washing, associated GTPases were visualized by autoradiography. (d) IRSp53 contains a partial CRIB motif, as shown in this alignment of the IRSp53 amino acid residues 268–280 with other Rac and Cdc42 effectors. Conserved residues are boxed
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3. Figure 2
IRSp53 induces filopodia and cell retraction. (a) Quiescent, subconfluent Swiss 3T3 cells were microinjected with (i) biotin dextran as control or with (ii) an expression vector encoding Myc-tagged full-length IRSp53. Cells were fixed 90 min after microinjection and stained for injection markers (data not shown), protein expression (data not shown), and filamentous actin (shown). (b) Quiescent, confluent Swiss 3T3 cells were injected as in (a), fixed 4.5 hr after injections, and stained with injection markers (data not shown) and for protein expression (shown). The scale bar represents 20 μm
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4. Figure 3
IRSp53 is regulated by an autoinhibitory N terminus. (a) Myc-tagged IRSp53 fragments (ΔNTΔCT or CT IRSp53) together with an empty vector or with Flag-tagged NT IRSp53 were transiently expressed in COS-7 cells. Lysates were subjected to immunoprecipitations using an antibody against the Myc epitope. Immunoprecipitated IRSp53 fragments were detected simultaneously by immunoblotting, using antibodies against both the Flag and the Myc epitope tags. Immunoprecipitations were from cells transfected as follows. Lane 1: Flag-NT IRSp53; lane 2: Myc-ΔNTΔCT IRSp53; lane 3: Myc-CT IRSp53; lane 4: Flag-NT IRSp53 and Myc-ΔNTΔCT; lane 5: Flag-NT IRSp53 and Myc-CT IRSp53. (b) Quiescent, subconfluent Swiss 3T3 cells were microinjected with an expression vector encoding (i and ii) Myc-tagged full-length IRSp53 or (iii and iv) full-length IRSp53 together with NT IRSp53. Cells were fixed 90 min after injection and stained to visualize epitope tags (data not shown) and filamentous actin (shown). Panels ii and iv represent 5-fold enlargements of i and iii, respectively. (c) Quiescent, confluent Swiss 3T3 cells were microinjected with an expression vector encoding (i and ii) Myc-tagged full-length IRSp53 or (iii and iv) full-length IRSp53 together with NT IRSp53. Cells were fixed 4.5 hr after injections and stained to visualize (i and iii) epitope tags and (ii and iv) filamentous actin. The scale bar represents 20 μm
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5. Figure 4
IRSp53 mediates Cdc42-induced formation of filopodia. Quiescent, subconfluent Swiss 3T3 cells were microinjected with an expression vector encoding (a,b) epitope-tagged FGD1 DH/PH, (c,d) FGD1 DH/PH and NT IRSp53, (e) Tiam1 DH/PH, or (f) Tiam1 DH/PH and NT IRSp53. Cells were fixed 90 min after injections and stained to visualize epitope tags (data not shown) and filamentous actin (shown). The scale bar represents 20 μm. The images in (b) and (d) are 7.5-fold enlargements of the images in (a) and (c), respectively
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6. Figure 5
IRSp53 interacts directly with Mena and Scar2. (a) In vitro-translated, [35S]Met Mena and Scar2 proteins (lanes 1 and 2) were allowed to interact with (lanes 3 and 5) recombinant GST or (lanes 4 and 6) GST-ΔNT IRSp53 fusion proteins immobilized on glutathione agarose. After extensive washing, proteins were resolved by SDS-PAGE; bound Mena and Scar2 proteins were visualized by autoradiography (lanes 3–6). (b) GST fusions of IRSp53 constructs were immobilized on nitrocellulose, as indicated on the right-hand side, and the blot was overlaid with in vitro-translated [35S]Met Mena. After washing, Mena that had associated with the IRSp53 truncations was visualized by autoradiography
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7. Figure 6
The N terminus and the CRIB motif of IRSp53 regulate Mena binding to the IRSp53 SH3 domain. (a) Myc-tagged ΔNT IRSp53 or Flag-tagged NT IRSp53 and Myc-ΔNT IRSp53 were transiently expressed in COS-7 cells, immunoprecipitated using anti-myc antibody-coated sepharose beads, and incubated with in vitro- translated [35S]Met Mena protein. To visualize bait proteins, the gel was stained with Coomassie blue (left panel) prior to exposing for autoradiography to visualize associated Mena (right panel). Molecular weight markers are indicated on the left-hand side. (b) Quiescent, confluent Swiss 3T3 cells were microinjected with expression vectors encoding AU-tagged Mena together with (i and ii) full-length, Myc-tagged IRSp53 or (iii and iv) the ΔCRIB IRSp53 point mutant. Cells were fixed 3.5 hr after injections and (i and iii) stained with anti-AU antibody to visualize Mena and with (ii and iv) anti-myc antibody to visualize IRSp53. The scale bar represents 20 μm
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8. Figure 7
Mena and IRSp53 synergize to promote filopodia formation. Quiescent, subconfluent Swiss 3T3 cells were microinjected with an expression vector encoding (a) epitope-tagged Mena, (b,g) Mena and full-length IRSp53, (c) IRSp53, (d) Mena and F428A, P429A IRSp53, (e) Mena, IRSp53, and NT IRSp53, or (f) Mena and ΔCRIB IRSp53. Cells were fixed (b–g) 90 min after injections and (a) 3.5 hr after injection and were stained to visualize epitope tags (data not shown) and filamentous actin (shown). The scale bar represents 20 μm. (g) is a 5-fold enlargement of (b)
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9. Figure 8
A schematic model for the regulation of IRSp53 and its interaction with regulation Cdc42 and Mena. In the resting state, the IRSp53 N terminus interacts with the central region of the molecule, and the SH3 domain is masked. In response to an appropriate stimulatory signal, GTP-loaded Cdc42 binds to the CRIB motif. This abrogates the autoinhibitory, intramolecular interaction and allows the SH3 domain to interact with IRSp53 effector proteins such as Mena
Current Biology  , DOI: ( /S (01) )